Methods for lowering blood glucose

ABSTRACT

The present invention provides a method for lowering blood glucose levels in an animal by transplanting a population of pancreatic endocrine precursor cells into an animal.

CROSS REFERENCE TO RELATED APPLICATIONS

The application is a continuation of U.S. patent application Ser. No.16/134,831, filed Sep. 18, 2018, which is a continuation of U.S. patentapplication Ser. No. 12/838,998, filed Jul. 19, 2010, issued as U.S.Pat. No. 10,076,544 on Sep. 18, 2018, which claims priority to U.S.Provisional Patent Application No. 61/226,923, filed Jul. 20, 2009. Theabove-listed applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention provides a method for lowering blood glucoselevels in an animal by transplanting a population of pancreaticendocrine precursor cells into an animal.

BACKGROUND

Advances in cell-replacement therapy for Type I diabetes mellitus and ashortage of transplantable islets of Langerhans have focused interest ondeveloping sources of insulin-producing cells, or P cells, appropriatefor engraftment. One approach is the generation of functional 3 cellsfrom pluripotent stem cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, a pluripotent cell gives rise to agroup of cells comprising three germ layers (ectoderm, mesoderm, andendoderm) in a process known as gastrulation. Tissues such as, forexample, thyroid, thymus, pancreas, gut, and liver, will develop fromthe endoderm, via an intermediate stage. The intermediate stage in thisprocess is the formation of definitive endoderm. Definitive endodermcells express a number of markers, such as, for example, HNF3 beta,GATA4, MIXL1, CXCR4 and SOX17.

Formation of the pancreas arises from the differentiation of definitiveendoderm into pancreatic endoderm. Cells of the pancreatic endodermexpress the pancreatic-duodenal homeobox gene, PDX1. In the absence ofPDX1, the pancreas fails to develop beyond the formation of ventral anddorsal buds. Thus, PDX1 expression marks a critical step in pancreaticorganogenesis. The mature pancreas contains, among other cell types,exocrine tissue and endocrine tissue. Exocrine and endocrine tissuesarise from the differentiation of pancreatic endoderm.

Cells bearing the features of islet cells have reportedly been derivedfrom embryonic cells of the mouse. For example, Lumelsky et al. (Science292:1389, 2001) report differentiation of mouse embryonic stem cells toinsulin-secreting structures similar to pancreatic islets. Soria et al.(Diabetes 49:157, 2000) report that insulin-secreting cells derived frommouse embryonic stem cells normalize glycemia in streptozotocin-induceddiabetic mice.

In one example, Hori et al. (PNAS 99: 16105, 2002) disclose thattreatment of mouse embryonic stem cells with inhibitors ofphosphoinositide 3-kinase (LY294002) produced cells that resembled 3cells.

In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports thegeneration of insulin-producing cells from mouse embryonic stem cellsconstitutively expressing Pax4.

Micallef et al. reports that retinoic acid can regulate the commitmentof embryonic stem cells to form PDX1 positive pancreatic endoderm.Retinoic acid is most effective at inducing PDX1 expression when addedto cultures at day four of embryonic stem cell differentiation during aperiod corresponding to the end of gastrulation in the embryo (Diabetes54:301, 2005).

Miyazaki et al. reports a mouse embryonic stem cell line over-expressingPdx1. Their results show that exogenous Pdx1 expression clearly enhancedthe expression of insulin, somatostatin, glucokinase, neurogenin3, p48,Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53:1030, 2004).

Skoudy et al. reports that activin A (a member of the TGF-β superfamily)upregulates the expression of exocrine pancreatic genes (p48 andamylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouseembryonic stem cells. The maximal effect was observed using InM activinA. They also observed that the expression level of insulin and Pdx1 mRNAwas not affected by retinoic acid; however, 3 nM FGF7 treatment resultedin an increased level of the transcript for Pdx1 (Biochem. J. 379: 749,2004).

Shiraki et al. studied the effects of growth factors that specificallyenhance differentiation of embryonic stem cells into PDX1 positivecells. They observed that TGF-β2 reproducibly yielded a higherproportion of PDX1 positive cells (Genes Cells. 2005 June; 10(6):503-16.).

Gordon et al. demonstrated the induction of brachyury [positive]/HNF3beta [positive] endoderm cells from mouse embryonic stem cells in theabsence of serum and in the presence of activin along with an inhibitorof Wnt signaling (US 2006/0003446A1).

Gordon et al. (PNAS, Vol 103, page 16806, 2006) states “Wnt andTGF-beta/nodal/activin signaling simultaneously were required for thegeneration of the anterior primitive streak”.

However, the mouse model of embryonic stem cell development may notexactly mimic the developmental program in higher mammals, such as, forexample, humans.

Thomson et al. isolated embryonic stem cells from human blastocysts(Science 282:114, 1998). Concurrently, Gearhart and coworkers derivedhuman embryonic germ (hEG) cell lines from fetal gonadal tissue(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlikemouse embryonic stem cells, which can be prevented from differentiatingsimply by culturing with Leukemia Inhibitory Factor (LIF), humanembryonic stem cells must be maintained under very special conditions(U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).

D'Amour et al. describes the production of enriched cultures of humanembryonic stem cell-derived definitive endoderm in the presence of ahigh concentration of activin and low serum (Nature Biotechnology 2005).Transplanting these cells under the kidney capsule of mice resulted indifferentiation into more mature cells with characteristics of someendodermal organs. Human embryonic stem cell-derived definitive endodermcells can be further differentiated into PDX1 positive cells afteraddition of FGF-10 (US 2005/0266554A1).

D'Amour et al. (Nature Biotechnology—24, 1392-1401 (2006)) states: “Wehave developed a differentiation process that converts human embryonicstem (hES) cells to endocrine cells capable of synthesizing thepancreatic hormones insulin, glucagon, somatostatin, pancreaticpolypeptide and ghrelin. This process mimics in vivo pancreaticorganogenesis by directing cells through stages resembling definitiveendoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursoren route to cells that express endocrine hormones”.

In another example, Fisk et al. reports a system for producingpancreatic islet cells from human embryonic stem cells(US2006/0040387A1). In this case, the differentiation pathway wasdivided into three stages. Human embryonic stem cells were firstdifferentiated to endoderm using a combination of sodium butyrate andactivin A. The cells were then cultured with TGF-β antagonists such asNoggin in combination with EGF or betacellulin to generate PDX1 positivecells. The terminal differentiation was induced by nicotinamide.

In one example, Benvenistry et al. states: “We conclude thatover-expression of PDX1 enhanced expression of pancreatic enrichedgenes, induction of insulin expression may require additional signalsthat are only present in vivo” (Benvenistry et al., Stem Cells 2006;24:1923-1930).

In another example, US2008/0241107A1 claims a method for producing acell that secretes insulin comprising: a) obtaining a cell that does notproduce insulin; and, b) incubating the cell with media containing highglucose, wherein the cell secretes insulin.

Therefore, there still remains a significant need to develop conditionsfor establishing pluripotent stem cell lines that can be expanded toaddress the current clinical needs, while retaining the potential todifferentiate into pancreatic endocrine cells, pancreatic hormoneexpressing cells, or pancreatic hormone secreting cells. We have takenan alternative approach to improve the efficiency of differentiatinghuman embryonic stem cells toward pancreatic endocrine cells.

SUMMARY

In one embodiment, the present invention provides a method for loweringblood glucose levels in an animal by transplanting a population ofpancreatic endocrine precursor cells into an animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the effect of different basal media on the expressionof NKX6.1 (FIG. 1A), PDX1 (FIG. 1B), PTF1 alpha (FIG. 1C) and NGN3 (FIG.1D). Duplicate samples were collected at stage 4, day 3 for real-timePCR analysis. The plots represent fold induction for each gene relativeto the DMEM/F12.

FIGS. 2A-2H show immunofluorescence images of the pancreatic marker PDX1(FIGS. 2A-2B), NKX6.1 (FIGS. 2C-2D), CDX2 (FIGS. 2E-2F) and NGN3 (FIGS.2G-2H) for cells treated with DMEM/F12 (FIGS. 2A, 2C, 2E, 2G) and cellstreated with DMEM-high glucose (FIGS. 2B, 2D, 2F, 2H) at stage 4 day 3,treated as described in Example 1.

FIGS. 3A-3F show the expression of PDX1 (FIG. 3A), NKX6.1 (FIG. 3B),PTF1 alpha (FIG. 3C), NGN3 (FIG. 3D), PAX4 (FIG. 3E) and NKX2.2 (FIG.3F) from samples of cells treated according to the methods described inExample 2. Duplicate samples were collected for real-time PCR analysisat the indicated times. The plots represent fold induction for each generelative to the expression of genes at stage 3 day 1.

FIG. 4 shows the expression of insulin (INS), glucagon (GCG), PDX1,NKX6.1, NGN3, MAFB and NEUROD in cells treated according to the methodsdescribed in Example 2. Duplicate samples were collected for real-timePCR analysis. The plots represent fold induction for each gene relativeto the expression of genes at stage 3 day 4. The light gray barsrepresent data from samples taken from cells harvested at stage 3 day 4.The dark gray bars represent data from samples taken from cellsharvested at stage 4 day 3. The black bars represent data from samplestaken from cells harvested at stage 5 day 5.

FIG. 5A shows the expression of PDX1, NKX6.1, NGN, and PTF1 alpha incells treated according to the methods described in Example 3. Duplicatesamples were collected for real-time PCR analysis at stage 4 day 3. Theplots represent fold induction for each gene relative to the expressionof genes of Treatment group one at stage 4 day 3. The light grey barsrepresent data from samples taken from cells harvested from the T1(treatment 1) group. The white bars represent data from samples takenfrom cells harvested from the T2 (treatment 2) group. The dark grey barsrepresent data from samples taken from cells harvested from the T3(treatment 3) group. The black bars represent data from samples takenfrom cells harvested from the T4 (treatment 4) group. FIG. 5B shows theexpression of insulin in cells treated according to the methodsdescribed in Example 3. Duplicate samples were collected for real-timePCR analysis at stage 4 day 3 (S4, D3), and at stage 4, day 8 (S4, D8).The plots represent fold induction for each gene relative to theexpression of genes of Treatment group one (T1) at stage 4 day 3.

FIG. 6 shows glucose stimulated human C-peptide release kinetics oftransplanted endocrine precursor cells. Specifically shown are thelevels of human C-peptide (y-axis) 60 minutes after glucoseadministration. The x-axis indicates the animal number and dayspost-transplant.

FIGS. 7A-7B show glucose stimulated of human C-peptide release kineticsof transplanted endocrine precursor cells. Specifically shown are thelevels of human C-peptide (y-axis) 60 minutes after glucoseadministration (FIG. 7A), and the levels of human C-peptide before andafter glucose administration (FIG. 7B). The x-axis indicates the animalnumber and days post-transplant.

FIG. 8 shows glucose stimulated of human C-peptide release kinetics oftransplanted endocrine precursor cells. Specifically shown are thelevels of human C-peptide (y-axis) 60 minutes after glucoseadministration. The x-axis indicates the animal number and dayspost-transplant.

FIGS. 9A-9B show glucose stimulated of human C-peptide release kineticsof transplanted endocrine precursor cells. Specifically shown are thelevels of human C-peptide (y-axis) 60 minutes after glucoseadministration (FIG. 9A), and the levels of human C-peptide before andafter glucose administration (FIG. 9B). The x-axis indicates the animalnumber and days post-transplant.

FIGS. 10A-10B shows glucose stimulated of human C-peptide releasekinetics of transplanted endocrine precursor cells. Specifically shownare the levels of human C-peptide (y-axis) 60 minutes after glucoseadministration (FIG. 10A), and the levels of human C-peptide before andafter glucose administration (FIG. 10B). The x-axis indicates the animalnumber and days post-transplant.

FIGS. 11A-11B shows the morphological and immunofluorescence analysis ofgraft samples 3 weeks post implant. (FIG. 11A) Micrographs from serialsections staining for human nuclear antigen and DAPI; (FIG. 11B)staining for CK19 and PDX1.

FIGS. 12A-12E show the morphological and immunofluorescence analysis ofgraft samples stained for insulin and glucagon at 3 weeks (FIG. 12A), 10weeks (FIG. 12B) and 13 weeks (FIG. 12C) post implant. FIG. 12D showsthe morphological and immunofluorescence analysis of graft samplesstained for PDX1 and insulin at 13 weeks post implant. FIG. 12E showsthe morphological and immunofluorescence analysis of graft samplesstained for NEUROD1 and insulin at 13 weeks post implant.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following subsectionsthat describe or illustrate certain features, embodiments orapplications of the present invention.

Definitions

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm), as well as to give rise to tissues of multiplegerm layers following transplantation and to contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent, meaning able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, meaning able to give rise toall embryonic cell types; (3) multipotent, meaning able to give rise toa subset of cell lineages but all within a particular tissue, organ, orphysiological system (for example, hematopoietic stem cells (HSC) canproduce progeny that include HSC (self-renewal), blood cell restrictedoligopotent progenitors, and all cell types and elements (e.g.,platelets) that are normal components of the blood); (4) oligopotent,meaning able to give rise to a more restricted subset of cell lineagesthan multipotent stem cells; and (5) unipotent, meaning able to giverise to a single cell lineage (e.g., spermatogenic stem cells).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cellsuch as, for example, a nerve cell or a muscle cell. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

“Cells expressing markers characteristic of the definitive endodermlineage”, or “Stage 1 cells”, or “Stage 1”, as used herein, refers tocells expressing at least one of the following markers: SOX-17, GATA4,HNF3 beta, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein,FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99,or OTX2. Cells expressing markers characteristic of the definitiveendoderm lineage include primitive streak precursor cells, primitivestreak cells, mesendoderm cells and definitive endoderm cells.

“Cells expressing markers characteristic of the pancreatic endodermlineage”, as used herein, refers to cells expressing at least one of thefollowing markers: PDX1, HNF-1 beta, PTF1 alpha, HNF6, or HB9. Cellsexpressing markers characteristic of the pancreatic endoderm lineageinclude pancreatic endoderm cells, primitive gut tube cells, andposterior foregut cells.

“Cells expressing markers characteristic of the pancreatic endocrinelineage”, as used herein, refers to cells expressing at least one of thefollowing markers: NEUROD, ISL1, PDX1, NKX6.1, MAFB, insulin, glucagon,or somatostatin. Cells expressing markers characteristic of thepancreatic endocrine lineage include pancreatic endocrine cells,pancreatic hormone expressing cells, and pancreatic hormone secretingcells, and cells of the B-cell lineage.

“Definitive endoderm”, as used herein, refers to cells which bear thecharacteristics of cells arising from the epiblast during gastrulationand which form the gastrointestinal tract and its derivatives.Definitive endoderm cells express the following markers: HNF3 beta,GATA4, SOX17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1.

“Markers”, as used herein, are nucleic acid or polypeptide moleculesthat are differentially expressed in a cell of interest. In thiscontext, differential expression means an increased level for a positivemarker and a decreased level for a negative marker. The detectable levelof the marker nucleic acid or polypeptide is sufficiently higher orlower in the cells of interest compared to other cells, such that thecell of interest can be identified and distinguished from other cellsusing any of a variety of methods known in the art.

“Pancreatic endocrine cell”, or “pancreatic hormone expressing cell”, asused herein, refers to a cell capable of expressing at least one of thefollowing hormones: insulin, glucagon, somatostatin, and pancreaticpolypeptide.

“Pancreatic endocrine precursor cell”, as used herein refers to amultipotent cell of the definitive endoderm lineage that expresses NGN3and which can further differentiate into cells of the endocrine systemincluding, but not limited to, pancreatic islet hormone-expressingcells. Endocrine precursor cells cannot differentiate into as manydifferent cell, tissue and/or organ types as compared to lessspecifically differentiated definitive endoderm lineage cells, such asPDX1 positive pancreatic endoderm cells.

“Pancreatic hormone producing cell”, as used herein, refers to a cellcapable of producing at least one of the following hormones: insulin,glucagon, somatostatin, and pancreatic polypeptide.

“Pancreatic hormone secreting cell” as used herein, refers to a cellcapable of secreting at least one of the following hormones: insulin,glucagon, somatostatin, and pancreatic polypeptide.

Isolation, Expansion and Culture of Pluripotent Stem CellsCharacterization of Pluripotent Stem Cells

Pluripotent stem cells may express one or more of the stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Differentiation of pluripotent stem cells in vitroresults in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (ifpresent) and increased expression of SSEA-1. Undifferentiatedpluripotent stem cells typically have alkaline phosphatase activity,which can be detected by fixing the cells with 4% paraformaldehyde, andthen developing with Vector Red as a substrate, as described by themanufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiatedpluripotent stem cells also typically express Oct-4 and TERT, asdetected by RT-PCR.

Another desirable phenotype of propagated pluripotent stem cells is apotential to differentiate into cells of all three germinal layers:endoderm, mesoderm, and ectoderm tissues. Pluripotency of pluripotentstem cells can be confirmed, for example, by injecting cells into severecombined immunodeficient (SCID) mice, fixing the teratomas that formusing 4% paraformaldehyde, and then examining them histologically forevidence of cell types from the three germ layers. Alternatively,pluripotency may be determined by the creation of embryoid bodies andassessing the embryoid bodies for the presence of markers associatedwith the three germinal layers.

Propagated pluripotent stem cell lines may be karyotyped using astandard G-banding technique and compared to published karyotypes of thecorresponding primate species. It is desirable to obtain cells that havea “normal karyotype,” which means that the cells are euploid, whereinall human chromosomes are present and not noticeably altered.

Sources of Pluripotent Stem Cells

The types of pluripotent stem cells that may be used include establishedlines of pluripotent cells derived from tissue formed after gestation,including pre-embryonic tissue (such as, for example, a blastocyst),embryonic tissue, or fetal tissue taken any time during gestation,typically but not necessarily before approximately 10-12 weeksgestation. Non-limiting examples are established lines of humanembryonic stem cells or human embryonic germ cells, such as, for examplethe human embryonic stem cell lines H1, H7, and H9 (WiCell). Alsocontemplated is use of the compositions of this disclosure during theinitial establishment or stabilization of such cells, in which case thesource cells would be primary pluripotent cells taken directly from thesource tissues. Also suitable are cells taken from a pluripotent stemcell population already cultured in the absence of feeder cells. Alsosuitable are mutant human embryonic stem cell lines, such as, forexample, BG01v (BresaGen, Athens, Ga.).

In one embodiment, human embryonic stem cells are prepared as describedby Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998;Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A.92:7844, 1995).

Culture of Pluripotent Stem Cells

In one embodiment, pluripotent stem cells are typically cultured on alayer of feeder cells that support the pluripotent stem cells in variousways. Alternatively, pluripotent stem cells are cultured in a culturesystem that is essentially free of feeder cells, but nonethelesssupports proliferation of pluripotent stem cells without undergoingsubstantial differentiation. The growth of pluripotent stem cells infeeder-free culture without differentiation is supported using a mediumconditioned by culturing previously with another cell type.Alternatively, the growth of pluripotent stem cells in feeder-freeculture without differentiation is supported using a chemically definedmedium.

For example, Reubinoff et al. (Nature Biotechnology 18: 399-404 (2000))and Thompson et al. (Science 6 Nov. 1998: Vol. 282. no. 5391, pp.1145-1147) disclose the culture of pluripotent stem cell lines fromhuman blastocysts using a mouse embryonic fibroblast feeder cell layer.

Richards et al., (Stem Cells 21: 546-556, 2003) evaluated a panel ofeleven different human adult, fetal and neonatal feeder cell layers fortheir ability to support human pluripotent stem cell culture. Richardset al., states: “human embryonic stem cell lines cultured on adult skinfibroblast feeders retain human embryonic stem cell morphology andremain pluripotent”.

US20020072117 discloses cell lines that produce media that support thegrowth of primate pluripotent stem cells in feeder-free culture. Thecell lines employed are mesenchymal and fibroblast-like cell linesobtained from embryonic tissue or differentiated from embryonic stemcells. US20020072117 also discloses the use of the cell lines as aprimary feeder cell layer.

In another example, Wang et al (Stem Cells 23: 1221-1227, 2005)discloses methods for the long-term growth of human pluripotent stemcells on feeder cell layers derived from human embryonic stem cells.

In another example, Stojkovic et al (Stem Cells 2005 23: 306-314, 2005)disclose a feeder cell system derived from the spontaneousdifferentiation of human embryonic stem cells.

In a further example, Miyamoto et al (Stem Cells 22: 433-440, 2004)disclose a source of feeder cells obtained from human placenta.

Amit et al (Biol. Reprod 68: 2150-2156, 2003) discloses a feeder celllayer derived from human foreskin.

In another example, Inzunza et al (Stem Cells 23: 544-549, 2005)disclose a feeder cell layer from human postnatal foreskin fibroblasts.

U.S. Pat. No. 6,642,048 discloses media that support the growth ofprimate pluripotent stem (pPS) cells in feeder-free culture, and celllines useful for production of such media. U.S. Pat. No. 6,642,048states: “This invention includes mesenchymal and fibroblast-like celllines obtained from embryonic tissue or differentiated from embryonicstem cells. Methods for deriving such cell lines, processing media, andgrowing stem cells using the conditioned media are described andillustrated in this disclosure.”

In another example, WO2005014799 discloses conditioned medium for themaintenance, proliferation and differentiation of mammalian cells.WO2005014799 states: “The culture medium produced in accordance with thepresent invention is conditioned by the cell secretion activity ofmurine cells; in particular, those differentiated and immortalizedtransgenic hepatocytes, named MMH (Met Murine Hepatocyte).”

In another example, Xu et al (Stem Cells 22: 972-980, 2004) disclosesconditioned medium obtained from human embryonic stem cell derivativesthat have been genetically modified to over express human telomerasereverse transcriptase.

In another example, US20070010011 discloses a chemically defined culturemedium for the maintenance of pluripotent stem cells.

An alternative culture system employs serum-free medium supplementedwith growth factors capable of promoting the proliferation of embryonicstem cells. For example, Cheon et al (BioReprod DOI:10.1095/biolreprod.105.046870, Oct. 19, 2005) disclose a feeder-free,serum-free culture system in which embryonic stem cells are maintainedin unconditioned serum replacement (SR) medium supplemented withdifferent growth factors capable of triggering embryonic stem cellself-renewal.

In another example, Levenstein et al (Stem Cells 24: 568-574, 2006)disclose methods for the long-term culture of human embryonic stem cellsin the absence of fibroblasts or conditioned medium, using mediasupplemented with bFGF.

In another example, US20050148070 discloses a method of culturing humanembryonic stem cells in defined media without serum and withoutfibroblast feeder cells, the method comprising: culturing the stem cellsin a culture medium containing albumin, amino acids, vitamins, minerals,at least one transferrin or transferrin substitute, at least one insulinor insulin substitute, the culture medium essentially free of mammalianfetal serum and containing at least about 100 ng/ml of a fibroblastgrowth factor capable of activating a fibroblast growth factor signalingreceptor, wherein the growth factor is supplied from a source other thanjust a fibroblast feeder layer, the medium supported the proliferationof stem cells in an undifferentiated state without feeder cells orconditioned medium.

In another example, US20050233446 discloses a defined media useful inculturing stem cells, including undifferentiated primate primordial stemcells. In solution, the media is substantially isotonic as compared tothe stem cells being cultured. In a given culture, the particular mediumcomprises a base medium and an amount of each of bFGF, insulin, andascorbic acid necessary to support substantially undifferentiated growthof the primordial stem cells.

In another example, U.S. Pat. No. 6,800,480 states “In one embodiment, acell culture medium for growing primate-derived primordial stem cells ina substantially undifferentiated state is provided which includes a lowosmotic pressure, low endotoxin basic medium that is effective tosupport the growth of primate-derived primordial stem cells. The basicmedium is combined with a nutrient serum effective to support the growthof primate-derived primordial stem cells and a substrate selected fromthe group consisting of feeder cells and an extracellular matrixcomponent derived from feeder cells. The medium further includesnon-essential amino acids, an anti-oxidant, and a first growth factorselected from the group consisting of nucleosides and a pyruvate salt.”

In another example, US20050244962 states: “In one aspect the inventionprovides a method of culturing primate embryonic stem cells. Onecultures the stem cells in a culture essentially free of mammalian fetalserum (preferably also essentially free of any animal serum) and in thepresence of fibroblast growth factor that is supplied from a sourceother than just a fibroblast feeder layer. In a preferred form, thefibroblast feeder layer, previously required to sustain a stem cellculture, is rendered unnecessary by the addition of sufficientfibroblast growth factor.”

In a further example, WO2005065354 discloses a defined, isotonic culturemedium that is essentially feeder-free and serum-free, comprising: a. abasal medium; b. an amount of bFGF sufficient to support growth ofsubstantially undifferentiated mammalian stem cells; c. an amount ofinsulin sufficient to support growth of substantially undifferentiatedmammalian stem cells; and d. an amount of ascorbic acid sufficient tosupport growth of substantially undifferentiated mammalian stem cells.

In another example, WO2005086845 discloses a method for maintenance ofan undifferentiated stem cell, said method comprising exposing a stemcell to a member of the transforming growth factor-beta (TGF-β3) familyof proteins, a member of the fibroblast growth factor (FGF) family ofproteins, or nicotinamide (NIC) in an amount sufficient to maintain thecell in an undifferentiated state for a sufficient amount of time toachieve a desired result.

The pluripotent stem cells may be plated onto a suitable culturesubstrate. In one embodiment, the suitable culture substrate is anextracellular matrix component, such as, for example, those derived frombasement membrane or that may form part of adhesion moleculereceptor-ligand couplings. In one embodiment, the suitable culturesubstrate is MATRIGEL® (Becton Dickenson). MATRIGEL® is a solublepreparation from Engelbreth-Holm Swarm tumor cells that gels at roomtemperature to form a reconstituted basement membrane.

Other extracellular matrix components and component mixtures aresuitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparan sulfate, and the like, alone or in variouscombinations.

The pluripotent stem cells may be plated onto the substrate in asuitable distribution and in the presence of a medium that promotes cellsurvival, propagation, and retention of the desirable characteristics.All these characteristics benefit from careful attention to the seedingdistribution and can readily be determined by one of skill in the art.

Suitable culture media may be made from the following components, suchas, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco#11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco#10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco#15039-027; non-essential amino acid solution, Gibco 11140-050;P3-mercaptoethanol, Sigma # M7522; human recombinant basic fibroblastgrowth factor (bFGF), Gibco #13256-029.

Formation of Pancreatic Endocrine Precursor Cells

In one embodiment, the present invention provides a method for producingpancreatic endocrine precursor cells, comprising the steps of:

-   -   a. Culturing pluripotent stem cells,    -   b. Differentiating the pluripotent stem cells into cells        expressing markers characteristic of the definitive endoderm        lineage,    -   c. Differentiating the cells expressing markers characteristic        of the definitive endoderm lineage into cells expressing markers        characteristic of the pancreatic endoderm lineage, and    -   d. Differentiating the cells expressing markers characteristic        of the pancreatic endoderm lineage into pancreatic endocrine        precursor cells.

Pluripotent stem cells suitable for use in the present inventioninclude, for example, the human embryonic stem cell line H9 (NIH code:WA09), the human embryonic stem cell line H1 (NIH code: WA01), the humanembryonic stem cell line H7 (NIH code: WA07), and the human embryonicstem cell line SA002 (Cellartis, Sweden). Also suitable for use in thepresent invention are cells that express at least one of the followingmarkers characteristic of pluripotent cells: ABCG2, CRIPTO, CD9, FOXD3,Connexin43, Connexin45, OCT4, SOX2, Nanog, hTERT, UTF1, ZFP42, SSEA-3,SSEA-4, Tra 1-60, Tra 1-81.

Markers characteristic of the definitive endoderm lineage are selectedfrom the group consisting of SOX17, GATA4, HNF3 beta, GSC, CER1, Nodal,FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin(EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable foruse in the present invention is a cell that expresses at least one ofthe markers characteristic of the definitive endoderm lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the definitive endoderm lineage is a primitive streakprecursor cell. In an alternate aspect, a cell expressing markerscharacteristic of the definitive endoderm lineage is a mesendoderm cell.In an alternate aspect, a cell expressing markers characteristic of thedefinitive endoderm lineage is a definitive endoderm cell.

Markers characteristic of the pancreatic endoderm lineage are selectedfrom the group consisting of PDX1, HNF1 beta, HNF6, HB9 and PROX1.Suitable for use in the present invention is a cell that expresses atleast one of the markers characteristic of the pancreatic endodermlineage. In one aspect of the present invention, a cell expressingmarkers characteristic of the pancreatic endoderm lineage is apancreatic endoderm cell.

Markers characteristic of pancreatic endocrine precursor cells areselected from the group consisting of NGN3, NKX6.1, NeuroD, ISL1, PDX1,PAX4, NKX2.2, or ARX. Suitable for use in the present invention is acell that expresses at least one of the markers characteristic ofpancreatic endocrine precursor cells.

Formation of Cells Expressing Markers Characteristic of the DefinitiveEndoderm Lineage

Pluripotent stem cells may be differentiated into cells expressingmarkers characteristic of the definitive endoderm lineage by any methodin the art or by any method proposed in this invention.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in D'Amour et al., NatureBiotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in Shinozaki et al., Development 131,1651-1662 (2004).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in McLean et al., Stem Cells 25,29-38 (2007).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in D'Amour et al., NatureBiotechnology 24, 1392-1401 (2006).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A inthe absence of serum, then culturing the cells with activin A and serum,and then culturing the cells with activin A and serum of a differentconcentration. An example of this method is disclosed in NatureBiotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A inthe absence of serum, then culturing the cells with activin A with serumof another concentration. An example of this method is disclosed inD'Amour et al., Nature Biotechnology, 2005.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A anda Wnt ligand in the absence of serum, then removing the Wnt ligand andculturing the cells with activin A with serum. An example of this methodis disclosed in Nature Biotechnology 24, 1392-1401 (2006).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 11/736,908, assigned to LifeScan,Inc.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 11/779,311, assigned to LifeScan,Inc.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 60/990,529.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,889.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,900.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,908.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,915.

Characterization of Cells Expressing Markers Characteristic of theDefinitive Endoderm Lineage

Formation of cells expressing markers characteristic of the definitiveendoderm lineage may be determined by testing for the presence of themarkers before and after following a particular protocol. Pluripotentstem cells typically do not express such markers. Thus, differentiationof pluripotent cells is detected when cells begin to express them.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by cells expressingmarkers characteristic of the definitive endoderm lineage.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998))

Characteristics of pluripotent stem cells are well known to thoseskilled in the art, and additional characteristics of pluripotent stemcells continue to be identified. Pluripotent stem cell markers include,for example, the expression of one or more of the following: ABCG2,CRIPTO, FOXD3, Connexin43, Connexin45, OCT4, SOX2, Nanog, hTERT, UTF1,ZFP42, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81.

After treating pluripotent stem cells with the methods of the presentinvention, the differentiated cells may be purified by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker, such as CXCR4, expressed bycells expressing markers characteristic of the definitive endodermlineage.

Formation of Cells Expressing Markers Characteristic of the PancreaticEndoderm Lineage from Cells Expressing Markers Characteristic of theDefinitive Endoderm Lineage

Cells expressing markers characteristic of the definitive endodermlineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endoderm lineage by any method in theart or by any method proposed in this invention.

For example, cells expressing markers characteristic of the definitiveendoderm lineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endoderm lineage according to themethods disclosed in D'Amour et al., Nature Biotechnology 24, 1392-1401(2006).

For example, cells expressing markers characteristic of the definitiveendoderm lineage are further differentiated into cells expressingmarkers characteristic of the pancreatic endoderm lineage, by treatingthe cells expressing markers characteristic of the definitive endodermlineage with a fibroblast growth factor and the hedgehog signalingpathway inhibitor KAAD-cyclopamine, then removing the medium containingthe fibroblast growth factor and KAAD-cyclopamine and subsequentlyculturing the cells in medium containing retinoic acid, a fibroblastgrowth factor and KAAD-cyclopamine. An example of this method isdisclosed in Nature Biotechnology 24, 1392-1401 (2006).

In one aspect of the present invention, cells expressing markerscharacteristic of the definitive endoderm lineage are furtherdifferentiated into cells expressing markers characteristic of thepancreatic endoderm lineage, by treating the cells expressing markerscharacteristic of the definitive endoderm lineage with retinoic acid andat least one fibroblast growth factor for a period of time, according tothe methods disclosed in U.S. patent application Ser. No. 11/736,908,assigned to LifeScan, Inc.

In one aspect of the present invention, cells expressing markerscharacteristic of the definitive endoderm lineage are furtherdifferentiated into cells expressing markers characteristic of thepancreatic endoderm lineage, by treating the cells expressing markerscharacteristic of the definitive endoderm lineage with retinoic acid andat least one fibroblast growth factor for a period of time, according tothe methods disclosed in U.S. patent application Ser. No. 11/779,311,assigned to LifeScan, Inc.

In one aspect of the present invention, cells expressing markerscharacteristic of the definitive endoderm lineage are furtherdifferentiated into cells expressing markers characteristic of thepancreatic endoderm lineage, by treating the cells expressing markerscharacteristic of the definitive endoderm lineage according to themethods disclosed in U.S. patent application Ser. No. 60/990,529.

Characterization of Cells Expressing Markers Characteristic of thePancreatic Endoderm Lineage

Markers characteristic of the pancreatic endoderm lineage are well knownto those skilled in the art, and additional markers characteristic ofthe pancreatic endoderm lineage continue to be identified. These markerscan be used to confirm that the cells treated in accordance with thepresent invention have differentiated to acquire the propertiescharacteristic of the pancreatic endoderm lineage. Pancreatic endodermlineage specific markers include the expression of one or moretranscription factors such as, for example, HLXB9, PTF1 alpha, PDX1,HNF6, HNF-1 beta.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by cells expressingmarkers characteristic of the pancreatic endoderm lineage.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)).

Formation of Pancreatic Endocrine Precursor Cells from Cells ExpressingMarkers Characteristic of the Pancreatic Endoderm Lineage

In one aspect of the present invention, cells expressing markerscharacteristic of the pancreatic endoderm lineage are differentiatedinto pancreatic endocrine precursor cells, by culturing the cellsexpressing markers characteristic of the pancreatic endoderm lineage inmedium supplemented with a factor capable of inhibiting BMP and a TGF-βreceptor I kinase inhibitor.

In one embodiment, the factor capable of inhibiting BMP is noggin.Noggin may be used at a concentration from about 100 pg/ml to about 500μg/ml. In one embodiment, noggin is used at a concentration of 100ng/ml.

In one embodiment, the TGF-β receptor I kinase inhibitor is ALK5inhibitor II (Calbiochem, Ca). ALK5 inhibitor II may be used at aconcentration from about 0.1 μM to about 10 μM. In one embodiment, ALK5inhibitor II is used at a concentration of 1 μM.

In one embodiment, the medium is DMEM containing 4500 mg/l glucose and1% B27.

In one embodiment, the cells are cultured in the culture medium forabout four days.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by pancreaticendocrine precursor cells.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)).

Characteristics of pluripotent stem cells are well known to thoseskilled in the art, and additional characteristics of pluripotent stemcells continue to be identified. Pluripotent stem cell markers include,for example, the expression of one or more of the following: ABCG2,CRIPTO, FOXD3, Connexin43, Connexin45, OCT4, SOX2, Nanog, hTERT, UTF1,ZFP42, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81.

After treating pluripotent stem cells with the methods of the presentinvention, the differentiated cells may be purified by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker, such as CXCR4, expressed bycells expressing markers characteristic of the pancreatic endodermlineage.

Markers characteristic of the pancreatic endoderm lineage are selectedfrom the group consisting of PDX1, HNF-1 beta, PTF1 alpha, HNF6, HB9 andPROX1. Suitable for use in the present invention is a cell thatexpresses at least one of the markers characteristic of the pancreaticendoderm lineage. In one aspect of the present invention, a cellexpressing markers characteristic of the pancreatic endoderm lineage isa pancreatic endoderm cell.

Markers characteristic of pancreatic endocrine precursor cells areselected from the group consisting of NGN3, NKX6.1, NEUROD, ISL1, PDX1,PAX4, NKX2.2, PAX6 or ARX.

Formation of Cells Expressing Markers Characteristic of the PancreaticEndocrine Lineage from Pancreatic Endocrine Precursor Cells

In one embodiment, pancreatic endocrine precursor cells, produced by themethods of the present invention may be further differentiated intocells expressing markers characteristic of the pancreatic endocrinelineage.

Pancreatic endocrine precursor cells may be differentiated into cellsexpressing markers characteristic of the pancreatic endocrine lineage byany method in the art or by any method proposed in this invention.

For example, pancreatic endocrine precursor cells obtained according tothe methods of the present invention are further differentiated intocells expressing markers characteristic of the pancreatic endocrinelineage, by culturing the pancreatic endocrine precursor cells in mediumcontaining exendin 4, then removing the medium containing exendin 4 andsubsequently culturing the cells in medium containing exendin 1, IGF1and HGF. An example of this method is disclosed in D' Amour et al.,Nature Biotechnology, 2006.

For example, pancreatic endocrine precursor cells obtained according tothe methods of the present invention are further differentiated intocells expressing markers characteristic of the pancreatic endocrinelineage, by culturing the pancreatic endocrine precursor cells in mediumcontaining DAPT (Sigma-Aldrich, MO) and exendin 4. An example of thismethod is disclosed in D' Amour et al., Nature Biotechnology, 2006.

For example, pancreatic endocrine precursor cells obtained according tothe methods of the present invention are further differentiated intocells expressing markers characteristic of the pancreatic endocrinelineage, by culturing the pancreatic endocrine precursor cells in mediumcontaining exendin 4. An example of this method is disclosed in D' Amouret al., Nature Biotechnology, 2006.

For example, cells pancreatic endocrine precursor cells obtainedaccording to the methods of the present invention are furtherdifferentiated into cells expressing markers characteristic of thepancreatic endocrine lineage, by treating the pancreatic endocrineprecursor cells with a factor that inhibits the Notch signaling pathway,according to the methods disclosed in U.S. patent application Ser. No.11/736,908, assigned to LifeScan, Inc.

For example, pancreatic endocrine precursor cells obtained according tothe methods of the present invention are further differentiated intocells expressing markers characteristic of the pancreatic endocrinelineage, by treating the pancreatic endocrine precursor cells with afactor that inhibits the Notch signaling pathway, according to themethods disclosed in U.S. patent application Ser. No. 11/779,311,assigned to LifeScan, Inc.

For example, pancreatic endocrine precursor cells obtained according tothe methods of the present invention are further differentiated intocells expressing markers characteristic of the pancreatic endocrinelineage, by treating the pancreatic endocrine precursor cells with afactor that inhibits the Notch signaling pathway, according to themethods disclosed in U.S. patent application Ser. No. 60/953,178,assigned to LifeScan, Inc.

For example, pancreatic endocrine precursor cells obtained according tothe methods of the present invention are further differentiated intocells expressing markers characteristic of the pancreatic endocrinelineage, by treating the pancreatic endocrine precursor cells with afactor that inhibits the Notch signaling pathway, according to themethods disclosed in U.S. patent application Ser. No. 60/990,529,assigned to LifeScan, Inc.

Markers characteristic of the pancreatic endocrine lineage are selectedfrom the group consisting of NEUROD, ISL1, PDX1, NKX6.1, PAX4, PAX6,NGN3, and NKX2.2. In one embodiment, a pancreatic endocrine cell iscapable of expressing at least one of the following hormones: insulin,glucagon, somatostatin, and pancreatic polypeptide. Suitable for use inthe present invention is a cell that expresses at least one of themarkers characteristic of the pancreatic endocrine lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the pancreatic endocrine lineage is a pancreaticendocrine cell. The pancreatic endocrine cell may be a pancreatichormone-expressing cell. Alternatively, the pancreatic endocrine cellmay be a pancreatic hormone-secreting cell.

In one aspect of the present invention, the pancreatic endocrine cell isa cell expressing markers characteristic of the β cell lineage. A cellexpressing markers characteristic of the β cell lineage expresses PDX1and at least one of the following transcription factors: NGN-3, NKX2.2,NKX6.1, NEUROD, ISL1, HNF3 beta, MAFA, PAX4, and PAX6. In one aspect ofthe present invention, a cell expressing markers characteristic of the βcell lineage is a β cell.

Therapies

In one aspect, the present invention provides a method for treating apatient suffering from, or at risk of developing, Type1 diabetes. In oneembodiment, the method involves culturing pluripotent stem cells,differentiating the pluripotent stem cells in vitro into a β-celllineage, and implanting the cells of a β-cell lineage into a patient. Inan alternate embodiment, the method involves culturing pluripotent stemcells, differentiating the pluripotent stem cells in vitro intopancreatic endocrine precursor cells, and implanting the pancreaticendocrine precursor cells into a patient.

In yet another aspect, this invention provides a method for treating apatient suffering from, or at risk of developing, Type 2 diabetes. Inone embodiment, the method involves culturing pluripotent stem cells,differentiating the pluripotent stem cells in vitro into a β-celllineage, and implanting the cells of a β-cell lineage into a patient. Inan alternate embodiment, the method involves culturing pluripotent stemcells, differentiating the pluripotent stem cells in vitro intopancreatic endocrine precursor cells, and implanting the pancreaticendocrine precursor cells into a patient.

If appropriate, the patient can be further treated with pharmaceuticalagents or bioactives that facilitate the survival and function of thetransplanted cells. These agents may include, for example, insulin,members of the TGF-β family, including TGF-β1, 2, and 3, bonemorphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13),fibroblast growth factors-1 and -2, platelet-derived growth factor-AA,and —BB, platelet rich plasma, insulin growth factor (IGF-I, II) growthdifferentiation factor (GDF-5, -6, -7, -8, -10, -15), vascularendothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin,among others. Other pharmaceutical compounds can include, for example,nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1 and 2mimetibody, Exendin-4, retinoic acid, parathyroid hormone, MAPKinhibitors, such as, for example, compounds disclosed in U.S. PublishedApplication 2004/0209901 and U.S. Published Application 2004/0132729.

The pluripotent stem cells may be differentiated into aninsulin-producing cell prior to transplantation into a recipient. In aspecific embodiment, the pluripotent stem cells are fully differentiatedinto β-cells, prior to transplantation into a recipient. Alternatively,the pluripotent stem cells may be transplanted into a recipient in anundifferentiated or partially differentiated state. Furtherdifferentiation may take place in the recipient.

Definitive endoderm cells or, alternatively, pancreatic endoderm cells,or, alternatively, β cells, may be implanted as dispersed cells orformed into clusters that may be infused into the hepatic portal vein.Alternatively, cells may be provided in biocompatible degradablepolymeric supports, porous non-degradable devices or encapsulated toprotect from host immune response. Cells may be implanted into anappropriate site in a recipient. The implantation sites include, forexample, the liver, natural pancreas, renal subcapsular space, omentum,peritoneum, subserosal space, intestine, stomach, or a subcutaneouspocket.

To enhance further differentiation, survival or activity of theimplanted cells, additional factors, such as growth factors,antioxidants or anti-inflammatory agents, can be administered before,simultaneously with, or after the administration of the cells. Incertain embodiments, growth factors are utilized to differentiate theadministered cells in vivo. These factors can be secreted by endogenouscells and exposed to the administered cells in situ. Implanted cells canbe induced to differentiate by any combination of endogenous andexogenously administered growth factors known in the art.

The amount of cells used in implantation depends on a number of variousfactors including the patient's condition and response to the therapy,and can be determined by one skilled in the art.

In one aspect, this invention provides a method for treating a patientsuffering from, or at risk of developing diabetes. This method involvesculturing pluripotent stem cells, differentiating the cultured cells invitro into a β-cell lineage, and incorporating the cells into athree-dimensional support. The cells can be maintained in vitro on thissupport prior to implantation into the patient. Alternatively, thesupport containing the cells can be directly implanted in the patientwithout additional in vitro culturing. The support can optionally beincorporated with at least one pharmaceutical agent that facilitates thesurvival and function of the transplanted cells.

Support materials suitable for use for purposes of the present inventioninclude tissue templates, conduits, barriers, and reservoirs useful fortissue repair. In particular, synthetic and natural materials in theform of foams, sponges, gels, hydrogels, textiles, and nonwovenstructures, which have been used in vitro and in vivo to reconstruct orregenerate biological tissue, as well as to deliver chemotactic agentsfor inducing tissue growth, are suitable for use in practicing themethods of the present invention. See, for example, the materialsdisclosed in U.S. Pat. Nos. 5,770,417, 6,022,743, 5,567,612, 5,759,830,6,626,950, 6,534,084, 6,306,424, 6,365,149, 6,599,323, 6,656,488, U.S.Published Application 2004/0062753 A1, U.S. Pat. Nos. 4,557,264 and6,333,029.

To form a support incorporated with a pharmaceutical agent, thepharmaceutical agent can be mixed with the polymer solution prior toforming the support. Alternatively, a pharmaceutical agent could becoated onto a fabricated support, preferably in the presence of apharmaceutical carrier. The pharmaceutical agent may be present as aliquid, a finely divided solid, or any other appropriate physical form.Alternatively, excipients may be added to the support to alter therelease rate of the pharmaceutical agent. In an alternate embodiment,the support is incorporated with at least one pharmaceutical compoundthat is an anti-inflammatory compound, such as, for example compoundsdisclosed in U.S. Pat. No. 6,509,369.

The support may be incorporated with at least one pharmaceuticalcompound that is an anti-apoptotic compound, such as, for example,compounds disclosed in U.S. Pat. No. 6,793,945.

The support may also be incorporated with at least one pharmaceuticalcompound that is an inhibitor of fibrosis, such as, for example,compounds disclosed in U.S. Pat. No. 6,331,298.

The support may also be incorporated with at least one pharmaceuticalcompound that is capable of enhancing angiogenesis, such as, forexample, compounds disclosed in U.S. Published Application 2004/0220393and U.S. Published Application 2004/0209901.

The support may also be incorporated with at least one pharmaceuticalcompound that is an immunosuppressive compound, such as, for example,compounds disclosed in U.S. Published Application 2004/0171623.

The support may also be incorporated with at least one pharmaceuticalcompound that is a growth factor, such as, for example, members of theTGF-β family, including TGF-β1, 2, and 3, bone morphogenic proteins(BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growthfactors-1 and -2, platelet-derived growth factor-AA, and —BB, plateletrich plasma, insulin growth factor (IGF-I, II) growth differentiationfactor (GDF-5, -6, -8, -10, -15), vascular endothelial cell-derivedgrowth factor (VEGF), pleiotrophin, endothelin, among others. Otherpharmaceutical compounds can include, for example, nicotinamide, hypoxiainducible factor 1-alpha, glucagon like peptide-I (GLP-1), GLP-1 andGLP-2 mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid,parathyroid hormone, tenascin-C, tropoelastin, thrombin-derivedpeptides, cathelicidins, defensins, laminin, biological peptidescontaining cell- and heparin-binding domains of adhesive extracellularmatrix proteins such as fibronectin and vitronectin, MAPK inhibitors,such as, for example, compounds disclosed in U.S. Published Application2004/0209901 and U.S. Published Application 2004/0132729.

The incorporation of the cells of the present invention into a scaffoldcan be achieved by the simple depositing of cells onto the scaffold.Cells can enter into the scaffold by simple diffusion (J. Pediatr. Surg.23 (1 Pt 2): 3-9 (1988)). Several other approaches have been developedto enhance the efficiency of cell seeding. For example, spinner flaskshave been used in seeding of chondrocytes onto polyglycolic acidscaffolds (Biotechnol. Prog. 14(2): 193-202 (1998)). Another approachfor seeding cells is the use of centrifugation, which yields minimumstress to the seeded cells and enhances seeding efficiency. For example,Yang et al. developed a cell seeding method (J. Biomed. Mater. Res.55(3): 379-86 (2001)), referred to as Centrifugational CellImmobilization (CCI).

The present invention is further illustrated, but not limited by, thefollowing examples.

EXAMPLES Example 1 Formation of a Population of Pancreatic EndocrinePrecursor Cells

Cells of the human embryonic stem cell line H1 were cultured onMATRIGEL-coated plates (1:30 dilution), and differentiated intopancreatic endocrine precursor cells using the following protocol:

-   -   a. RPMI medium (Catalogue #22400, Invitrogen, Ca) supplemented        with 2% BSA (Catalog #152401, MP Biomedical, Ohio), and 100        ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog        #1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog        #100-18B, PeproTech, NJ), for one day followed by treatment with        RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus        8 ng/ml of bFGF for an additional two days (Stage 1), then    -   b. DMEM/F12 (Catalogue #11330, Invitrogen, Ca)+2% BSA+50 ng/ml        FGF7 for three days (Stage 2), then    -   c. Different basal media indicated in Table 1 were used,        supplemented with 1% B27 (#17504-044, Invitrogen, CA)+50 ng/ml        FGF7+0.25 μM Cyclopamine-KAAD (#239804, Calbiochem, CA)+2 μM        Retinoic acid (RA) (Sigma, MO)+100 ng/ml of Noggin (R & D        Systems, MN) for four days (Stage 3), then    -   d. Different basal media indicated in Table 1 were used,        supplemented with 1% B27 (Invitrogen, CA)+100 ng/ml Noggin+1 M        ALK5 inhibitor II (Catalog #616452, Calbiochem, Ca) for three        days (Stage 4).

TABLE 1 Basal Media Basal Media Catalogue# (Stage 3) (Stage 4)(Invitrogen, CA) Treatment 1 DMEM DMEM 11995-040 (High Glucose) (HighGlucose) Treatment 2 DMEM DMEM 10567-014 (Low Glucose) (Low Glucose)Treatment 3 CMRL CMRL 11530-037 Treatment 4 DMEM/F12 DMEM/F12 11039-021

Cultures were sampled in duplicate at stage four day three ofdifferentiation and analyzed for expression of pancreatic markers usingreal-time PCR. In parallel, stage four, day three cultures were fixedand stained for the following proteins: NKX6.1 (Catalogue # F64A6B4,Developmental Studies Hybridoma Bank, University of Iowa), PDX1, NGN3,and CDX2.

Stage four, day three samples, cultured in DMEM medium (treatment 1 andtreatment 2, Table 1), showed significant increases in the level ofNKX6.1, NGN3 and PTF1 alpha expression by PCR (FIG. 1) compared to cellscultured in DMEM/F12 (treatment 4, table 1) or CMRL (treatment 3, Table1). No differences in the level of PDX1 expression was observed in thecultures tested. However, immunocytochemistry revealed that cellscultured in DMEM/F12 medium, a large proportion of PDX1 expressing cellsalso expressed CDX2, a marker for gut endoderm (FIGS. 2A, 2E). Incontrast, the cells treated in DMEM medium provided a separation of PDX1positive cells and CDX2 positive cells (FIGS. 2B, 2F), wherein a largeproportion of PDX1 expressing cells did not express CDX2.

In addition, cells expressing PDX1 that were obtained from cells treatedin DMEM also expressed NKX6.1. As seen in FIG. 2, 50 to 60% of the PDX1positive cells also expressed NKX6.1 by the end of stage 4 (FIG. 2D) and20 to 30% of the PDX1 positive cells expressed NGN3 (FIG. 2H). However,co-expression of NKX6.1 and NGN3 was not observed in cells cultured inDMEM. The co-expression of PDX1 and NGN3 was also observed in cellscultured in DMEM/F12 or CMRL medium (FIG. 2G), however, the expressionof NKX6.1 was not observed in cells treated in either DMEM/F12 or CMRLmedium (FIG. 2C).

These data suggest that different basal medium facilitate the generationof different pancreatic endoderm cell populations: By using DMEM/F12, apopulation that co-expresses PDX1 and CDX2 was generated, while the useof DMEM resulted in a population that expressed PDX1 and NKX6.1, but didnot express CDX2. Further, the data suggests that the expression ofpancreatic gene expression was increased by increasing the glucoseconcentration of the culture medium. See FIG. 1 and FIG. 2

Example 2 Direct Differentiation of Human Embryonic Stem Cells toPancreatic Endocrine Precursor Cells

Cells of the human embryonic stem cell line H1 were cultured onMATRIGEL-coated plates (1:30 dilution), and differentiated intopancreatic endocrine precursor cells using the following protocol:

-   -   a. RPMI medium (Catalogue #22400, Invitrogen, Ca) supplemented        with 2% BSA (Catalog #152401, MP Biomedical, Ohio), and 100        ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog        #1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog        #100-18B, PeproTech, NJ), for one day followed by treatment with        RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus        8 ng/ml of bFGF for an additional two days (Stage 1), then    -   b. DMEM/F12 (Catalogue #11330, Invitrogen, Ca)+2% BSA+50 ng/ml        FGF7 for three days (Stage 2), then    -   c. DMEM (high glucose)+1% B27 (Invitrogen, CA)+50 ng/ml        FGF7+0.25 μM Cyclopamine-KAAD+2 μM Retinoic acid (RA) (Sigma,        MO)+100 ng/ml of Noggin (R & D Systems, MN) for four days (Stage        3), then    -   d. DMEM (high glucose)+1% B27 (Invitrogen, CA)+100 ng/ml        Noggin+1 μM ALK5 inhibitor II (Catalog #616452, Calbiochem, Ca)        for three days (Stage 4), then    -   e. DMEM (high glucose)+0.5% ITS (Invitrogen, CA)+0.1% BSA+1 μM        Alk5 inhibitor II+100 ng/ml Noggin+20 ng/ml Betacellulin (R&D        Systems, MN) for five days (Stage 5).

Cultures were sampled in duplicate each day from stage 2 day 3 to stagefour day 3 of differentiation and analyzed for expression of pancreaticmarkers using real-time PCR. After the cells entered stage 4, a dramaticincrease of PDX1, NKX6.1 and PTF1 alpha was observed (FIGS. 3A-3C). Inaddition, a significant up-regulation of NGN3, PAX4, NKX2.2 and NEURODwas also observed (FIGS. 3D-3F). PAX4, NKX2.2 and NEUROD, are directlyregulated by NGN3, which suggests that the pancreatic endoderm initiatedthe commitment to the pancreatic endocrine lineage.

Further differentiation of the pancreatic endocrine precursor cells invitro into insulin expressing cells was achieved by the addition of aTGF-3 receptor I kinase inhibitor, Noggin and Betacellulin. As shown inFIG. 4, a significant increase in insulin expression was observedfollowing the addition of Alk5 inhibitor II (a TGF-3 receptor I kinaseinhibitor), Noggin and Betacellulin for five days. NGN3 and PAX4expression levels declined, while the level of expression of PDX1,NKX6.1 MAFB and NEUROD remained constant.

Example 3 An Alternate Method for the Direct Differentiation of HumanEmbryonic Stem Cells to Pancreatic Endocrine Precursor Cells

This example demonstrates an alternative method for differentiatinghuman embryonic stem cells to pancreatic endocrine precursors using Alk5inhibitor II (an inhibitor of TGF-beta receptor family), together with alow dose of exogenous retinoid, for example retinol (vitamin A), whichmay be present in media supplements such as B27.

Cells of the human embryonic stem cell line H1 at passages 45 werecultured on MATRIGEL-coated plates (1:30 dilution), and differentiatedinto pancreatic endocrine precursor cells using the following protocol:

-   -   a. RPMI medium supplemented with 2% BSA, and 100 ng/ml activin A        plus 20 ng/ml WNT-3a plus 8 ng/ml of bFGF, for one day followed        by treatment with RPMI media supplemented with 2% BSA and 100        ng/ml activin A plus 8 ng/ml of bFGF for an additional two days        (Stage 1), then    -   b. DMEM/F12+2% BSA+50 ng/ml FGF7 for three days (Stage 2), then    -   c. DMEM (high glucose)+1% B27 (Invitrogen, CA)+50 ng/ml        FGF7+0.25 aM Cyclopamine-KAAD+0.1 μM Retinoic acid (RA)+100        ng/ml Noggin for four days (Treatmentl, Stage 3), or    -   d. DMEM (high glucose)+1% B27 (Invitrogen, CA)+50 ng/ml        FGF7+0.25 μM Cyclopamine-KAAD+0.1 μM Retinoic acid (RA)+1 M Alk5        inhibitor+Noggin 100 ng/ml for four days (Treatment 2, Stage 3),        or    -   e. DMEM (high glucose)+1% B27 (Invitrogen, CA)+50 ng/ml        FGF7+0.25 μM Cyclopamine-KAAD+1 M Alk5 inhibitor+100 ng/ml        Noggin for four days (Treatment 3, Stage 3), or    -   f. DMEM (high glucose)+1% B27 (Invitrogen, CA)+50 ng/ml        FGF7+0.25 μM Cyclopamine-KAAD+2 μM Retinoic acid (RA)+100 ng/ml        Noggin for four days (Treatment 4, Stage 3), then    -   DMEM (high glucose)+1% B27 (Invitrogen, CA)+100 ng/ml Noggin+1 M        ALK5 inhibitor II for eight days (Stage 4).

Cultures were sampled in duplicate on day 3 and day 8 of stage 4 ofdifferentiation, and analyzed for expression of pancreatic markers usingreal-time PCR.

Treatment of cells expressing markers characteristic of the pancreaticendoderm lineage in medium supplemented with FGF7, Noggin andCyclopamine-KAAD, ALK5 inhibitor II and with either low dose retinoicacid (0.1 M) or no exogenous retinoic acid, induced the expression ofNGN3 and continued up regulation of PDX1 and NKX6.1 (FIG. 5A, Treatment3 and 4). The level of expression of NGN3 was similar in cells treatedwith a high dose (2 μM) of retinoic acid (FIG. 5A, Treatment 4respectively). These data suggest that the addition of Alk5 inhibitor IIis sufficient to induce the formation of pancreatic endocrine progenitorcells, when cells expressing markers characteristic of the pancreaticendoderm lineage are treated with FGF7, Noggin and Cyclopamine-KAAD. SeeFIG. 5A, where no expression of NGN3 was observed in cells treated witha low dose of retinoic acid (0.1 M) in the absence of Alk5 inhibitor II(FIG. 5A, Treatment 1). The pancreatic endocrine cells formed by theabove treatment were competent to form insulin expressing cells invitro. See FIG. 5B, wherein the NGN3 expressing cells formed expressedinsulin following treatment with DMEM (high glucose)+1% B27 (Invitrogen,CA)+100 ng/ml Noggin+1 M ALK5 inhibitor II for eight days.

Example 4 In Vivo Maturation of Pancreatic Endocrine Precursor Cells

Cells of the human embryonic stem cell line H1 at passages 45 werecultured on MATRIGEL-coated plates (1:30 dilution), and differentiatedinto pancreatic endocrine precursor cells using the following protocol:

-   -   a. RPMI medium+2% BSA+100 ng/ml activin A+20 ng/ml WNT-3a+8        ng/ml of bFGF for one day followed by treatment with RPMI        media+2% BSA+100 ng/ml activin A+8 ng/ml of bFGF for an        additional two days (Stage 1), then    -   b. DMEM/F12+2% BSA+50 ng/ml FGF7 for three days (Stage 2), then    -   c. DMEM-High glucose+1% B27+50 ng/ml FGF7+0.25 μM        Cyclopamine-KAAD+2 μM Retinoic acid (RA)+100 ng/ml of Noggin for        four days (Stage 3), then    -   d. DMEM-High glucose+1% B27+100 ng/ml Noggin+1 M ALK5 inhibitor        II for three days (Stage 4).

The above method (method 1) of culturing the cells in vitro was used forthe transplantations in Animal Nos. 8, 11, 14, 17, 20 and 23. See FIG.6.

An alternate differentiation protocol was also tested, wherein cells ofthe human embryonic stem cell line H1 at passages 45 were cultured onMATRIGEL-coated plates (1:30 dilution), and differentiated intopancreatic endocrine precursor cells using the following protocol:

-   -   a. RPMI medium+2% BSA+100 ng/ml activin A+20 ng/ml WNT-3a+8        ng/ml of bFGF for one day followed by treatment with RPMI        media+2% BSA+100 ng/ml activin A+8 ng/ml of bFGF for an        additional two days (Stage 1), then    -   b. DMEM/F12+2% BSA+50 ng/ml FGF7 for three days (Stage 2), then    -   c. DMEM(high-glucose)+1% B27+50 ng/ml FGF7+0.25 μM        Cyclopamine-KAAD+2 μM Retinoic acid (RA)+100 ng/ml of        Noggin+Alk5 inhibitor II 1 μM for four days (stage 3), then    -   d. DMEM (high-glucose)+1% B27+100 ng/ml Noggin+Alk5 inhibitor II        1 M for three days (stage 4).

Cells at the end of stage four were mechanically scored using a 1-mlglass pipette and subsequently transferred to non-adherent plates forculture overnight. The resultant aggregates were collected, andaggregates, containing 5 to 8 million cells were transplanted into thekidney capsule of an immuno-compromised mice (SCID/Bg) mouse. Thismethod (method 2) of culturing the cells in vitro was used for thetransplantations in Animal Nos. 324,326,329,331,333. See FIGS. 7A-7B.

An alternate differentiation protocol was also tested, wherein cells ofthe human embryonic stem cell line H1 at passages 45 were cultured onMATRIGEL-coated plates (1:30 dilution), and differentiated intopancreatic endocrine precursor cells using the following protocol:

-   -   a. RPMI medium+2% BSA+100 ng/ml activin A+20 ng/ml WNT-3a+8        ng/ml of bFGF for one day followed by treatment with RPMI        media+2% BSA+100 ng/ml activin A+8 ng/ml of bFGF for an        additional two days (Stage 1), then    -   b. DMEM/F12+2% BSA+50 ng/ml FGF7 for three days (Stage 2), then    -   c. Culturing the cells for four days in DMEM(high-glucose)+1%        B27+50 ng/ml FGF7+0.25 μM Cyclopamine-KAAD+0.1 μM Retinoic acid        (RA)+100 ng/ml of Noggin+Alk5 inhibitor II 1 M (stage 3), then    -   d. DMEM (high-glucose)+1% B27 for three days (stage 4).

Cells at the end of stage four were mechanically scored using a 1-mlglass pipette and subsequently transferred to non-adherent plates forculture overnight. The resultant aggregates were collected, andaggregates, containing 5 to 8 million cells were transplanted into thekidney capsule of an immuno-compromised mice (SCID/Bg) mouse. Thismethod (method 3) of culturing the cells in vitro was used for thetransplantations in Animal Nos. 294, 295, 296, 297. See FIG. 8.

An alternate differentiation protocol was also tested, wherein cells ofthe human embryonic stem cell line H1 at passages 45 were cultured onMATRIGEL-coated plates (1:30 dilution), and differentiated intopancreatic endocrine precursor cells using the following protocol:

-   -   a. RPMI medium+2% BSA+100 ng/ml activin A+20 ng/ml WNT-3a+8        ng/ml of bFGF for one day followed by treatment with RPMI        media+2% BSA+100 ng/ml activin A+8 ng/ml of bFGF for an        additional two days (Stage 1), then    -   b. DMEM/F12+2% BSA+50 ng/ml FGF7 for three days (Stage 2), then    -   c. Culturing the cells for four days in DMEM(high-glucose)+1%        B27+50 ng/ml FGF7+0.25 μM Cyclopamine-KAAD+0.1 μM Retinoic acid        (RA)+100 ng/ml of Noggin+Alk5 inhibitor II 1 M (stage 3), then    -   d. DMEM (high-glucose)+1% B27+100 ng/ml of Noggin+Alk5 inhibitor        II 1 μM for three days (stage 4).

Cells at the end of stage four were mechanically scored using a 1-mlglass pipette and subsequently transferred to non-adherent plates forculture overnight. The resultant aggregates were collected, andaggregates, containing 5 to 8 million cells were transplanted into thekidney capsule of an immuno-compromised mice (SCID/Bg) mouse. Thismethod (method 4) of culturing the cells in vitro was used for thetransplantations in Animal Nos. 336, 338, 340, 342, 344. See FIGS.9A-9B.

An alternate differentiation protocol was also tested, wherein cells ofthe human embryonic stem cell line H1 at passages 45 were cultured onMATRIGEL-coated plates (1:30 dilution), and differentiated intopancreatic endocrine precursor cells using the following protocol:

-   -   a. RPMI medium+2% BSA+100 ng/ml activin A+20 ng/ml WNT-3a+8        ng/ml of bFGF for one day followed by treatment with RPMI        media+2% BSA+100 ng/ml activin A+8 ng/ml of bFGF for an        additional two days (Stage 1), then    -   b. DMEM/F12+2% BSA+50 ng/ml FGF7 for three days (Stage 2), then    -   c. Culturing the cells for four days in DMEM(high-glucose)+1%        B27+50 ng/ml FGF7+0.25 μM Cyclopamine-KAAD+100 ng/ml of        Noggin+Alk5 inhibitor II 1 μM (stage 3), then    -   d. DMEM (high-glucose)+1% B27+100 ng/ml Noggin+Alk5 inhibitor II        (stage 4).

Cells at the end of stage four were mechanically scored using a 1-mlglass pipette and subsequently transferred to non-adherent plates forculture overnight. The resultant aggregates were collected, andaggregates, containing 5 to 8 million cells were transplanted into thekidney capsule of an immuno-compromised mice (SCID/Bg) mouse. Thismethod (method 5) of culturing the cells in vitro was used for thetransplantations in Animal Nos. 335,337,339,341,343. See FIGS. 10A-10B.

Five to six-week-old male scid-beige mice(C.B-Igh-1b/GbmsTac-Prkdc^(scid)-Lyst^(bg) N7) were purchased fromTaconic Farms. Mice were housed in microisolator cages with free accessto sterilized food and water. In preparation for surgery, mice wereidentified by ear tagging and their body weight measured and their bloodglucose determine by a hand held glucometer (One Touch, LifeScan).

Mice were anesthetized with a mixture of isoflurane and oxygen and thesurgical site was shaved with small animal clippers. Mice were dosedwith 0.1 mg·kg Buprenex subcutaneously pre-operatively. The surgicalsite was prepared with successive washes of 70% isopropyl alcohol and10% povidone-iodide.

Cells at the end of stage four were briefly treated with 1 mg/ml dispasefor five minutes and mechanically scored using a 1-ml glass pipette andsubsequently transferred to non-adherent plates for culture overnight.During the preoperative preparation of the mice, the cells werecentrifuged in a 1.5 ml microfuge tube and most of the supernatantremoved, leaving just enough to collect the pellet of cells. The cellswere collected into a Rainin Pos-D positive displacement pipette and thepipette was inverted to allow for the cells to settle by gravity. Theexcess media was dispensed leaving a packed cell preparation fortransplant.

For transplantation, a 24G×¾″ I.V. catheter was used to penetrate thekidney capsule and the needle was removed. The catheter was thenadvanced under the kidney capsule to the distal pole of the kidney. ThePos-D pipette tip was placed firmly in the hub of the catheter and the 5million cells dispensed from the pipette through the catheter under thekidney capsule and delivered to the distal pole of the kidney. Thekidney capsule was sealed with a low temperature cautery and the kidneywas returned its original anatomical position. In parallel, cellaggregates containing 5 million cells were loaded into the 50-μl deviceusing Post-D pipette tip. The 50-μl devices were purchased fromTheraCyte, Inc (Irvine, Calif.). The device was sealed by medicaladhesive silicone type A (Dow Corning, Cat #129109) after the loading,and implanted subcutaneously into SICD/Bg mice (animal Nos. 3 and 4).The muscle was closed with continuous sutures using 5-0 vicryl and theskin closed with wound clips. Mice were dosed with 1.0 mg·kg Metacamsubcutaneously post-operatively. The mouse was removed from theanesthesia and allowed to fully recover.

Following transplantation, mice were weighed once per week and bloodglucose measured twice a week. At various intervals followingtransplantation, mice were dosed with 3 g/kg glucose IP and blood drawnvia the retro-orbital sinus 60 minutes following glucose injection intomicrofuge tubes containing a small amount of heparin. The blood wascentrifuged and the plasma placed into a second microfuge tube andfrozen on dry ice and then stored at −80° C. until human c-peptide assaywas performed. Human c-peptide levels were determined using theMercodia/ALPCO Diagnostics Ultrasensitive C-peptide ELISA (Cat No.80-CPTHU-E01, Alpco Diagnostics, NH) according to the manufacturer'sinstructions.

Human C-peptide was detected in animal serum as early as 4 weeks aftertransplantation and increased over time. By the end of three months, theanimals were fasted for about 15-20 hrs, after which a blood sample(pre-glucose) was withdrawn retro-orbitally. Each animal then receivedan intraperitoneal injection dose of about 3 g/kg of glucose in 30%dextrose solution, and blood was withdrawn at about 60 minutes postglucose infusion. The serum was separated from the blood cells throughcentrifugation in micro-containers. The ELISA analysis was performed onduplicated 25 μl of serum using an ultra-sensitive human specificC-peptide ELISA plates (Cat No. 80-CPTHU-E01, Alpco Diagnostics, NH).The detection of human C-peptide indicates that insulin secretion isderived from the grafted cells.

Low serum levels of human C-peptide (less than 0.5 ng/ml) were detectedin response to glucose stimulation in all animals that received graftscontaining pancreatic endocrine precursor cells, 60 days aftertransplantation. Between two to three months post-transplantation,glucose-stimulated human serum level increased rapidly in those animals(FIGS. 6-10). In general, those animals receiving cell cluster graftsalso responded to glucose (FIGS. 7B, 9B and 10B).

Histological examination of grafts harvested at different time pointsrevealed the presence of human cells under the mice kidney capsule (asdetected using human nuclear antigen staining). See FIGS. 11A-11B. Cellstransplanted under the kidney capsule were observed to form duct-likestructures three weeks after implantation. See FIG. 11A. The number ofthe duct-like structures increased with time. Most of the duct-likestructures contained high levels of PDX1 and CK19 (FIG. 11B). Thissuggests that the pancreatic endocrine precursor cells were capable ofdifferentiating further in vivo.

Since PDX1 expression in ducts is important for specifying progenitorpopulations that eventually form the endocrine pancreas, theco-expression of PDX1 with either insulin or glucagon in the grafts wasdetermined. Insulin and glucagon expressing cells were observed ingrafts as early as three weeks post-transplant (FIG. 12A). Most ofendocrine hormonal expressing cells were formed when the PDX1 cellsmigrated out of the duct structure. A significant number of insulinpositive cells were detected in the graft around 10-week time point;most of the insulin positive cells expressed PDX1 and NKX6.1 (FIG. 12B).These data correlate with the C-peptide expression date reported above.At 20 weeks, the number of insulin positive cells increasedsignificantly, such that significant numbers of single cells, solelyexpressing insulin were detected in the graft. Most insulin expressingcells also expressed PDX1 (FIG. 12C), NKX6.1 (FIG. 12B), and NEUROD(FIG. 12E). PDX1 and NKX6.1 have been reported to be beneficial to themaintenance of glucose-stimulated insulin release of mature beta cells.

In a separate control experiment, animals received cells that weredifferentiated to the end of stage four in DMEM/F12, according to themethods described in Example 1. No human C-peptide was observed in theserum of any of the animals that received the cells, for up to threemonths post transplantation. Further, PCR and immunohistochemistryanalysis did not reveal the expression of insulin, PDX1 or NKX6.1.However, a significant amount of glucagon positive cells were observedin the graft three month after transplantation.

Example 5 Histological Examination of Grafts

Histological examination of grafts harvested from animals receivingtransplants were preformed substantially as described in previousexample.

The grafts from the animals treated in the previous example, weredissected from the animals and washed with PBS−/− (not containing Mg++and Ca++, Invitrogen) twice, and then transferred to 4%paraformaldehyde/PBS and fixed for about 2-3 hours at 4° C., and the PBS(−) was changed after 1 hour. The grafts were then equilibrated in 30%sucrose/PBS (−) overnight at 4° C. and mounted into OCT compound(SAKURA, #4583) and frozen with dry ice. The graft tissues were cut into10 micron sections using a cryostat, and sections were stored at −80° C.

For analysis, the frozen sections were allowed to thaw to roomtemperature, and once thawed the sections were washed with PBS 2 times,in Shandon slide cassettes. The tissue sections were permeabilized withPBS+0.5% Triton-X for 20 minutes, followed by a 2 ml PBS wash. Thesections were then incubated with a blocking solution containing 4%chicken serum/PBS. The slides were incubated for about 1 hour at roomtemperature. The blocking solution was removed away by 3 washes with 2ml PBS. The sections were incubated again in the Shandon slide cassettesovernight at 4° C. with the primary antibodies, which were diluted in 4%chicken serum.

After incubation with the primary antibodies the sides were then washedwith 2 ml PBS three times. The sections were again incubated in theShandon slide cassettes at room temperature with the appropriatesecondary antibodies, diluted in 4% chicken serum. After about 30 min toone hour, the sections were washed with 2 ml PBS 3 times before theywere removed from the Shandon slide cassettes and mounted withVectashield containing DAPI. Additional antibodies to other markerstypical of pancreatic hormone secreting cells were analyzed includingtranscription factors PDX1. See Table 2 below.

TABLE 2 Antibodies to pancreatic hormones and transcription factorsAntibody Host Dilution Provider Insulin Rabbit 1:100 Cell Signaling PDX1Goat 1:100 Santa Cruz Glucagon Mouse 1:100 Sigma CK19 Mouse 1:100 Dako

Publications cited throughout this document are hereby incorporated byreference in their entirety. Although the various aspects of theinvention have been illustrated above by reference to examples andpreferred embodiments, it will be appreciated that the scope of theinvention is defined not by the foregoing description but by thefollowing claims properly construed under principles of patent law.

What is claimed is:
 1. A method for lowering blood glucose levels in a mammal, comprising transplanting a population of pancreatic endocrine precursor cells expressing insulin, NKX6.1 and PDX1, but not expressing CDX2, into the liver, natural pancreas, renal subcapsular space, omentum, peritoneum, subserosal space, intestine, or a subcutaneous pocket of the mammal, wherein the pancreatic endocrine precursor cells express insulin, lower the blood glucose levels in the mammal, and are protected from host immune response when transplanted.
 2. The method of claim 1, wherein the pancreatic endocrine precursor cells are in a biocompatible degradable polymeric support, a porous non-degradable device or encapsulated when transplanted.
 3. The method of claim 2, wherein pancreatic endocrine precursor cells are encapsulated.
 4. The method of claim 3, wherein the encapsulated cells are transplanted subcutaneously.
 5. A method for lowering blood glucose levels in a mammal comprising: (a) differentiating pancreatic endocrine precursor cells expressing NKX6.1 and PDX1, but not expressing CDX2 to obtain a population of cells expressing insulin; and (b) transplanting the population of insulin expressing cells of step (a) into the liver, natural pancreas, renal subcapsular space, omentum, peritoneum, subserosal space, intestine, or a subcutaneous pocket of the mammal, wherein the pancreatic endocrine precursor cells express insulin, lower the blood glucose levels in the mammal, and are protected from host immune response when transplanted.
 6. The method of claim 5, wherein the insulin expressing cells are in a biocompatible degradable polymeric support, a porous non-degradable device or encapsulated when transplanted.
 7. The method of claim 6, wherein the cells are encapsulated.
 8. The method of claim 7, wherein the encapsulated cells are transplanted subcutaneously. 